Vitamina A is essential for the continued growth and general health of higher animals, including man. The vitamin is needed for vision, reproduction, and the maintenance of differentiated epithelia. With the exception of its role in the visual cycle, the mechanism of action of vitamin A is unknown. The involvement of the vitamin in cell growth and differentiation may ultimately explain the effects of the retinoids (vitamin A and its natural and synthetic derivatives) in preventing or reversing certain neoplasms). The research proposed here is aimed at determining in a rigorous and quantitative way, the subcellular localization of vitamin A in mammalian target epithelial tissues and in liver. The four tissues to be studied are intestinal epithelium, corneal epithelium, testes, and liver. Two model systems will be used: tissues isolated from intact rats and in vitro cultures of these cell types derived from rats. Cell homogenates will be fractionated by differential and isopycnic centrifugation and the fractions analyzed for retinol and its metabolites and for biochemical market constituents. A quantitative analytical approach will be used. Specific aims are: (1) To develop or adapt methods for the subcellular fractionation of these cell types including the establishment of appropriate markers enzymes for various cell organelles and membranes. (2) To define the distribution of vitamin A among the subcellular fractions using sensitive fluorometric or radioisotopic assays. (3) To determine the intracellular localization of vitamin A by comparison of its distribution with those of appropriate markers. (4) To obtain quantitative information on the molecular forms of the vitamin in the various tissues and fractions (i.e., to determine if the vitamin is present as free retinol, as retinyl esters, or as other metabolites and to assess if the vitamin is free or bound to macromolecules such as the intracellular binding proteins). (5) To determine the quantity and subcellular localization of the various molecular forms of the vitamin during progressive vitamin A depletion. Knowledge of the intracellular localization of vitamin A in target tissues may shed light on the biochemical mechanisms of action of this essential nutrient.